Systematic Review of Methods for Measuring Circulating Cell-Free DNA in Plasma of Healthy Individuals

Background/Objectives: Standardizing measurement of circulating cell-free DNA (cfDNA) in healthy individuals is critical for its application as a reference in biomarker research, yet methodological variability remains poorly documented. Methods: We systematically reviewed 35 studies (n = 1250 healthy subjects) assessing how pre-analytical handling, extraction kits, and quantification methods influence plasma cfDNA levels. We identified quantification approaches (qPCR vs. fluorometry) and use of custom extraction kits as the strongest drivers of variability. Results: In qPCR studies, including ≥ 40 subjects reduced variability, underscoring the importance of adequate sample size. Commercial kits produced more consistent yields than in-house protocols; in our dataset, many studies used Qiagen's QIAamp Circulating Nucleic Acid Kit, which has historically served as a widely used reference platform. Blood collection in EDTA tubes had minimal impact when commercial kits were used. Conclusions: Based on these findings, we recommend EDTA tubes, a standardized commercial extraction kit, and qPCR quantification to minimize cfDNA measurement variability in healthy cohorts. Finally, we provide expected cfDNA ranges for healthy individuals based on methodological flow, which can guide future benchmarking efforts and biomarker studies, improving comparability and early-detection research.